Volume 16, Issue 19, Pages 1938-1943 (October 2006) Microtubule-Associated AIR9 Recognizes the Cortical Division Site at Preprophase and Cell-Plate Insertion  Henrik Buschmann, Jordi Chan, Luis Sanchez-Pulido, Miguel A. Andrade-Navarro, John H. Doonan, Clive W. Lloyd  Current Biology  Volume 16, Issue 19, Pages 1938-1943 (October 2006) DOI: 10.1016/j.cub.2006.08.028 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 Polyacrylamide Gels of Arabidopsis Proteins Copurifying with Microtubules and Computational Analysis of a Novel Repeated Domain Found in AIR9 (A) Addition of taxol results in polymerization of endogenous tubulin (arrowhead). Lane 1 = no taxol, and lane 2 = plus taxol. (B) Same loading as in (A) but analyzed on low percentage gels (5%). High-molecular-weight bands enriched in B, lane 2, were cut out and analyzed by MALDI. Peptide fingerprints revealed the bands to correspond with: one asterisk = MOR1; two asterisks = RNA-helicase (At5g61140), see [5]; and one arrow = AIR9 (At2g34680). (C) Multiple sequence alignment of the repeated A9 domain. The sequences correspond to 11 domains in Arabidopsis AIR9 (At_AIR9), five in the Leishmania major AIR9 homolog (Lm_AIR9-like; UniProt:Q4Q4X1), and five in a bacterial sequence from Nocardiodes sp. JS614 (Nc_Q3GZG5). Coloring indicates amino acid conservation (BLOSUM62): cyan (>3), green (3–1.2), and magenta (1.2–0.4). Lanes below the alignment: secondary-structure prediction with PhD [21] for the A9-repeated domain with β-strands indicated by arrows (2D_PhD_Pred) and sequence (pdb1vca) and secondary X-Ray-determined structure (2D_1vca) of the second immunoglobulin domain of human VCAM-1 [22]. The green line marks a putative disulphide bridge for the A9 domains from Nocardiodes. Beta strands are labeled according to standard names in the immunoglobulin fold [10]. (D) Ribbon representation of a homology model of the third A9 domain of the Arabidopsis AIR9 protein with the structure of the second immunoglobulin domain of the human VCAM-1 protein [22] (see Supplemental Experimental Procedures). The C′ strand (yellow) was the region modeled with the lowest reliability. The main hydrophobic core of the domain is formed around the aromatic residue Trp-726. The conserved Asp-752 is hydrogen-bonded to the backbone nitrogen of Thr-749, capping a small 3–10 helix [23] typically located in the E-F loop of immunoglobulin domains [24]. Current Biology 2006 16, 1938-1943DOI: (10.1016/j.cub.2006.08.028) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 GFP-AIR9 Localizes to Cortical Microtubules of Interphase Cells, Labels the Site of Cell-Plate Insertion, and Marks an Inwardly Growing Torus on the New Cross-Wall (A) Cortical microtubules of an interphase BY-2 cell. (B) Two PPBs labeled by GFP-AIR9 (asterisks). (C) GFP-AIR9 shows weaker, diffuse labeling of the metaphase spindle. (D) During cytokinesis, GFP-AIR9 labels the phragmoplast but not the forming cell plate at the midline. (E) Polarized cytokinesis. A part of the phragmoplast is still present (left); GFP-AIR9 additionally labels the site of cell-plate insertion on the opposite side (right, arrowhead). (F) Surface view shows that labeling of the insertion site (is) directly follows the path of phragmoplast (ph) disassembly. (G) After cell-plate insertion, GFP-AIR9 enters the new cross-wall. (H) Projection of a divided cell with the cross-wall labeled by GFP-AIR9 (3 min after complete phragmoplast disassembly). No cortical microtubules can be seen at this stage; daughter nuclei (n) are indicated. (I) Control line expressing GFP-tubulin at the same stage as (H). (J) Control line expressing GFP-MBD at the same stage as (H). (K) After inward growth of the AIR9 torus, the cross-wall becomes evenly labeled but shows no filaments (rotated projection). (L) Later, the signal under the same cross-wall (as in [K]) became filamentous as it codistributed with nascent cortical microtubules. All micrographs are projections of confocal z sections, except (D)–(G), which are single z sections. Scales bars in (A), (B), (C), (D), (E), (F), and scale bars in (H)–(J) represent 20 μm; scale bars in (G), (K), and (L) represent 10 μm. Current Biology 2006 16, 1938-1943DOI: (10.1016/j.cub.2006.08.028) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 Microtubule Binding and Localization to the Cell-Plate-Insertion Site Are Conferred by Separate Parts of AIR9 (A) Transient expression of truncation constructs in Arabidopsis suspension cells. Fragments localizing to microtubules are in green; the GFP-Δ15 fragment in red showed cross-wall labeling. Expression of GFP-Δ16 (left) and GFP-Δ15 (right). AIR9 fragments are as follows: Δ1 (amino acids 1–478), Δ2 (amino acids 449–1195), Δ3 (amino acids 1161–1708), Δ6 (amino acids 244–478), Δ9 (amino acids 1–265), Δ10 (amino acids 1–105), Δ11 (amino acids 211–265), Δ12 (amino acids 1–234), Δ14 (amino acids 106–265), Δ15 (amino acids 244–1708), and Δ16 (amino acids 106–210). (B–D) Analysis of cytokinesis in stable BY-2 lines. (B) Z section of a GFP-Δ15 cell during phragmoplast disassembly revealing an inward-moving cross-wall signal (arrowhead); corresponding phase contrast picture (right) and the partial phragmoplast is indicated (ph). (C) The same cell as in (B) but a few minutes later; projection of z sections. Like full-length AIR9, GFP-Δ15 labels the insertion site and the cross-wall. (D) A projection of a BY-2 cell expressing GFP-Δ12 (which includes the microtubule binding site) a few minutes after phragmoplast disassembly. Arrowheads indicate position of the new cell wall. Inset shows microtubules of the same line during interphase. Scale bars in (A) represent 10 μm, and scale bars in (B)–(D) represent 20 μm. Current Biology 2006 16, 1938-1943DOI: (10.1016/j.cub.2006.08.028) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 Aberrantly Inserted Cell Plates Do Not Show an AIR9-Torus and Are Delayed in Maturation CIPC creates convoluted phragmoplasts and cell-plate insertions outside the former PPB zone. (A) PPB stage (0–4 min) and phragmoplast stages (84–99 min). CIPC-induced aberrant phragmoplasts show cell-plate insertions at predicted (asterisk and arrow) and ectopic (arrowhead) sites. (B) Quantitative evaluation of signal strength at insertion sites shown in (A). (C–H) Staining of cell-wall material with Calcofluor White for cellulose (C–E) and with aniline blue for callose (F–H). Control cells (C and F), and CIPC-treated cells 6 hr after wash-out (D and G) and 24 hr after wash-out (E and H). Scale bars in (A) represent 10 μm; scale bars in (C) and (F) represent 40 μm; and scale bars in (D), (E), (G), and (H) represent 20 μm. Current Biology 2006 16, 1938-1943DOI: (10.1016/j.cub.2006.08.028) Copyright © 2006 Elsevier Ltd Terms and Conditions